The present invention relates to IgE-dependent histamine-releasing factor (hereinafter, abbreviated as xe2x80x9cHRFxe2x80x9d) receptor, HRF-binding peptides and nucleic acids encoding the same, and uses thereof. More specifically, the present invention relates to novel receptors against HRF causing allergic diseases such as asthma, rhinitis, urticaria, anaphylaxis, allergic bronchiectasis, allergies due to foods, drugs, pollen, insects, etc., hay fever, cold urticaria or atopic dermatitis, HRF-binding peptides and nucleic acid encoding the same, and uses thereof in the medicinal area.
Allergies are known as being caused by inheritable hypersensitive formation of IgE in response to allergens, or disruption of balance between IL-4 (Interleukin-4) increasing IgE secretion and interferon decreasing IgE secretion. Upon the exposure to allergens, an immediate reaction occurs and various cells associated with inflammation are gathered, and after several hours, late-phase reaction (hereinafter, abbreviated as xe2x80x9cLPRxe2x80x9d) occurs by histamine and other cytokines secreted from basophils, eosinophils and lymphocytes. In LPR, histamine is secreted from basophils, but allergens, which have initiated the reaction, do not exist any longer. Further, LPR is developed in only about half of patients suffered from allergies. Therefore, what causes histamine secretion from basophils and what causes development into LPR have been issues of great interest. To the present time, cytokines such as MCP-3, MCP-1 or RANTES were known as secreting histamine. But, it was found that in IgE-dependent LPR, only HRF can induce histamine secretion from basophils (MacDonald, et al., 1995), the mechanism of which has never been known.
HRF, which is a ubiquitous cytoplasmic protein, is a known protein consisting of 172 amino acids (Bohm, et al., 1989). 45 Amino acids at its C-terminal form basic domain. Because such domain has about 46% homology with MAP-1B, microtubule-associated protein, it was assumed that HRF is also microtubule-associated protein. Gachet, et al. (1997) observed that HRF is distributed consistently with the cytoskeletal network to some extent by using confocal microscope, which suggests that HRF binds to the cytoskeleton. Meanwhile, Sanchez, et al. (1997) published that HRF, even though it does not fall within general Ca2+-binding protein family, binds to Ca2+ and further, identified that yeast cells can survive with the deletion of HRF genes in Saccharomyces cerevisiae. These suggest that HRF falls within the gene family having redundant pathway. MacDonald, et al. (1995) also found HRF, which is an intracellular protein, in the outside of cells. Further, it was known that HRF present in the outside of cells stimulates IgE-sensitized basophils to release histamine, but an accurate interaction between IgE and HRF has not been identified (Schroeder, et al., 1996). Schroeder, et al. (1997) observed that HRF can augment the anti-IgE-induced histamine release from all basophils, regardless of the IgE type, and thus suggested that HRF exerts its function by binding to cell membrane receptors, not by binding with IgE. Accordingly, the followings have been important issues, i.e. how HRF is secreted to the outside of cells and how it stimulates IgE-sensitized basophils to release histamine. Since HRF, a hydrophilic and intracellular protein, is detected in LPR allergy patients plasma at a large amount, it was assumed to be secreted to the outside of cells by apoptosis or other mechanisms and to release histamine via HRF receptors present in basophil membrane. In addition, because this HRF exists in most of tissues, it is assumed to function in tissue cells other than in inflammatory cells. But, its functions in other tissues than inflammatory cells, particularly in cerebral tissue or nerve cells, have never been reported. Recently, HRF was found during the analysis of proteins present in human brain using 2-D gel electrophoresis and proteomics (Langen, et al., 1999). Subsequently, it was also reported that HRF protein is decreased in the brain of patients died of Down""s syndrome or Alzheimer""s disease (Kim, et al., 2001).
On the other hand, (Na,K)ATPase, which involves in the formation of resting membrane potential and in the balanced regulation of osmosis within cells, is also present in nerve cells, particularly nerve end or synaptosomal membranes, at a high concentration and plays an important role in neuroactivity. It was reported that in case of inhibition or loss of (Na,K)ATPase activity in nerve cell membrane, various neuropathological changes or apoptosis occurs (Lees, 1991). This is also related to the report that the intracellular ATP essential for (Na,K)ATPase activity is rapidly exhausted in cerebral ischemia or anoxia state (Martin, et al., 1994; Santos, et al., 1996). Therefore, it is believed that this enzyme activity is also inhibited in such cerebral disease states. Moreover, it was confirmed that in rat brain tissue slices, synaptosomes and in vitro culture system, in case (Na,K)ATPase activity is inhibited, neurotransmitters release is increased. From other in vivo and in vitro studies, it was suggested that neurotransmitters release is increased in ischemia or anoxia-like conditions and the resulting activation of postsynaptic cell membrane receptors is an important procedure in nerve injury (Choi, 1990; Martin, et al., 1994).
Cerebral (Na,K)ATPase activity is regulated not only by neurotransmitters such as dopamine, serotonin, norepinephrine, glutamate, etc. but also by endogenous substances such as insulin, nitric oxide (NO), etc. An endogenous (Na,K)ATPase inhibitor named xe2x80x9cbrain ouabainxe2x80x9d, which is structurally similar to ouabain, glycoside extracted from plants, was identified (Budzikowski, et al., 1998). But, Rodriguez, et al. (1992) reported that there exists an endogenous ouabain-like factor specifically inhibiting (Na,K)ATPase activity in soluble brain fractions and having the different structure and properties from ouabain. They also reported that it blocks high affinity 3H-ouabain binding to induce neurotransmitters release, and involves in (Na,K)ATPase activity regulation by neurotransmitters as well. Recently, that substance was named endobain E (Vatta, et al., 1999), bur has not yet been identified.
Surprisingly, the present inventors found that HRF, even though it is a hydrophilic protein, can transit the cell membrane and HRF receptor corresponds to a third cytoplasmic domain (CD3) of (Na,K)ATPase by yeast two-hybrid assay. In addition, the inventors first identified an accurate mechanism by which extracellularly secreted HRF stimulates histamine release within basophils.
Further, on the basis of the results as described above, the inventors anticipated that any allergic diseases can be effectively prevented or treated by blocking HRF introduction into the cells and/or HRF binding with (Na,K)ATPase to inhibit histamine release. Therefore, they have performed extensive studies on peptides binding to HRF by screening 12 mer and 7 mer phage display libraries and as a result, obtained peptides of the specific sequences which can inhibit histamine secretion at a remarkably high rate and thus, completed the present invention.
Accordingly, an object of the present invention is to provide novel HRF receptors, peptide binding to HRF and uses thereof.
A first aspect of this invention relates to a rat HRF receptor having the amino acid sequence selected from SEQ ID No. 1, 2 or 3.
A second aspect thereof relates to a human HRF receptor having the amino acid sequence selected from SEQ ID No. 4, 5 or 6.
A third aspect thereof relates to a HRF receptor having the sequence homology of 85% or more with any one of the above amino acid sequences.
The HRF receptor may be a large cytoplasmic loop [CD (cytoplasmic domain) 3] of (Na,K)ATPase xcex11, xcex12 or xcex13 subunit.
A fourth aspect of this invention relates to a nucleic acid encoding any one of the above HRF receptors. The nucleic acid may have the nucleotide sequence selected from SEQ ID No. 7, 8 or 9 (rat HRF), or selected from SEQ ID No. 10, 11 or 12 (human HRF).
A fifth aspect thereof relates to a recombinant vector comprising the above nucleic acid.
A sixth aspect thereof relates to a cell transformed with the above vector.
A seventh aspect thereof relates to a screening method of HRF receptor-interactive compounds, which comprises contacting the transformed cells with test compounds and compounds known as interacting with the receptors, and then, selecting compounds decreasing the interaction of the known compounds from the test compounds (competition binding assay).
A eighth aspect of the present invention relates to a HRF-binding peptide having the amino acid sequence as represented by the following formula:
(A, L or W)-X-X-X-X-(A, L, S or W)-(A, P or M), 
wherein X represents any amino acid.
Preferably, the HRF-binding peptide in accordance with the invention has the amino acid sequence (A, L or W)-X-X-(Y, P or A)-(P, G or K)-(A, L, S or W)-(A, P or M).
More preferably, it has the amino acid sequence (A, L or W)-(V, Y, E or A)-(T, V, F or A)-(Y, P or A)-(P, G or K)-(A, L, S or W)-(A, P or M), exemplified by any one of SEQ ID Nos. 13 to 22.
Still more preferably, it has the amino acid sequence (A or W)-(Y or A)-(V or A)-(Y or A)-(P or K)-(S or A)-(M or A), for example, of SEQ ID No. 14, 16, 17, 18, 19, 20, 21 or 22.
Most preferably, it has the amino acid sequence W-(Y or A)-(V or A)-(Y or A)-(P or K)-(S or A)-M, for example, of SEQ ID No. 14, 17, 18, 19, 20 or 21.
Such RF-binding peptide may be composed of L-, D-, or L- and D-amino acids, and contain one or more modified amino acids, for example, amino acid derivatives or alkylated, particularly methylated, amino acids.
A ninth aspect of the present invention relates to a nucleic acid encoding the HRF binding peptide.
A tenth aspect thereof relates to a recombinant vector comprising the nucleic acid.
A eleventh aspect thereof relates to a cell transformed with the recombinant vector.
A twelfth aspect thereof relates to a composition for diagnosis, prophylaxis or treatment of allergies, particularly asthma, rhinitis, urticaria, anaphylaxis, allergic bronchiectasis, allergies due to foods, drugs, pollen, insects, etc., hay fever, cold urticaria, or atopic dermatitis. The composition comprises as an active ingredient the HRF-binding peptide or the nucleic acid encoding the same.
A thirteenth aspect thereof relates to an agent inducing the release of neurotransmitters, e.g. dopamine, comprising as an active ingredient HRF or the nucleic acid encoding the same.
A fourteenth aspect thereof relates to an agent inhibiting the release of neurotransmitters, e.g. dopamine, in particular, for diagnosis, prophylaxis or treatment of apoptosis-associated nerve diseases such as cerebral apoplexy, Alzheimer""s disease or Parkinson""s disease. The agent comprises as an active ingredient the BRF-binding peptide or the nucleic acid encoding the same.
A fifteenth aspect thereof relates to a composition for diagnosis, prophylaxis or treatment of malaria, comprising as an active ingredient the HRF-binding peptide or the nucleic acid encoding the same.
Hereinafter, the present invention will be explained in detail.
The present inventors first identified that HRF binds to large cytoplasmic loop (CD3) in (Na,K)ATPase xcex1 subunit by using yeast 2-hydrid assay. The inventors also found that HRF interacts with CD3 in (Na,K)ATPase xcex1 subunit by coimmunoprecipitation in yeast and mammalian cells and measured their binding affinity. Further, they confirmed that HRF receptor is CD3 in (Na,K)ATPase xcex1 subunit under confocal microscope.
Additionally, they demonstrated that HRF, a water-soluble protein, can enter the cells by confocal microscope and Western blotting, and identified that it increases the intracellular Na+ and Ca2+ concentrations and thus, the extracellular Ca2+ sources are consumed by Na/Ca exchanger. They also found that in the presence of IgE, BRF generates ROS (reactive oxygen species), which results in the inflow of much more Ca2+ to the cells.
From the above-described facts, an accurate mechanism by which HRF stimulates histamine release from basophils has been revealed. That is, extracellularly secreted HRF enters the basophils and binds to CD3 of (Na,K)ATPase xcex1 subunit and then, inhibits (Na,K)ATPase activity like ouabain thereby to increase intracellular Na+ and Ca2+ concentrations following the activation of Na/Ca exchanger. Further, in the presence of IgE, intracellular Ca2+ is further increased due to the generation of ROS, which ultimately stimulates histamine release. This means that HRF receptor is CD 3 of (Na,K)ATPase and HRF is a cytoplasmic repressor of (Na,K)ATPase.
Accordingly, the identity of HRF receptor was first revealed by the present inventors and thus, in the present invention, provided is a HRF receptor having the amino acid sequence of SEQ ID No 1, 2 or 3. This receptor corresponds to CD3 in xcex11, xcex12 or xcex13 subunit of (Na,K)ATPase isolated from rat. But, as long as the fact that HRF receptor corresponds to CD3 in a subunit of (Na,K)ATPase (Na,K)ATPase has been discovered by the present inventors, any person having an ordinary skill in the art can easily identify human HRF receptors. Therefore, human HRF receptors also fall within the scope of the present invention, which have the amino acid sequence of SEQ ID No 4 (xcex11), 5 (xcex12) or 6 (xcex13).
In rat (Na,K)ATPase, the sequence homology between xcex11 and xcex12 CD3 is 87.6%, and that between xcex12 and xcex13 CD3 is 89.4%. In CD3 of (Na,K)ATPase xcex1 subunits, the sequence homology between rat and human is 97.5% in xcex11, 99.3% in xcex12 and 98.8% in xcex13, respectively (see FIGS. 18 to 21). Accordingly, the present invention provides HRF receptors having the sequence homology of 85% or more with any of the amino acid sequences of SEQ ID Nos. as set forth above.
This invention provides nucleic acids encoding the HRF receptors, for example, having the nucleotide sequences selected from any one of SEQ ID Nos. 7 (rat xcex11), 8 (rat xcex12) and 9(rat xcex13), or any one of SEQ ID Nos. 10 (human xcex11), 11 (human xcex12) and 12 (human xcex13). In addition, provided are recombinant vectors comprising the above-described nucleic acids and cells transformed with the recombinant vectors as well.
This invention also provides a screening method of compounds interacting with HRF receptor, characterized by using the above-described cells in competition binding analysis. In the competition binding assay, the cells transformed with the recombinant vector containing the nucleic acid encoding HRF receptor and HRF protein are contacted with test compounds and compounds, which were already known as interacting with the receptor. Then, compounds which inhibit the interaction of the known compounds are selected among the above test compounds. The above method enables the screening of novel compounds, which can effectively regulate histamine release within cells.
Moreover, the invention provides peptides inhibiting histamine release by binding to HRF with a high specific affinity. In one embodiment, provided are peptides having the amino acid sequence of the following formula:
(A, L or W)-X-X-X-X-(A, L, S or W)-(A, P or M), 
wherein X represents any amino acid.
Examples of HRF-binding peptides include the followings:
(A, L or W)-X-X-(Y, P or A)-(P, G or K)-(A, L, S or W)-(A, P or M);
particularly, (A, L or W)-(V, Y, E or A)-(T, V, F or A)-(Y, P or A)-(P, G or K)-(A, L, S or W)-(A, P or M), e.g. SEQ ID Nos. 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22;
more particularly, (A or W)-(Y or A)-(V or A)-(Y or A)-(P or K)-(S or A)-(M or A), e.g. SEQ ID Nos. 14, 16, 17, 18, 19, 20, 21 or 22; and,
most particularly, W-(Y or A)-(V or A)-(Y or A)-(P or K)-(S or A)-M, e.g. SEQ ID Nos. 14, 17, 18, 19, 20 or 21.
The present inventors obtained the above peptides by phage displayed library screening and then, repeated experiments using synthetic peptides. The peptides in accordance with this invention may be chemically synthesized or prepared using genetic recombination technology. Preferably, domains composing the peptides may be prepared from proteins in vivo or parts thereof. The peptides may be prepared by recombinant DNA technology using expression vectors to which DNA encoding the peptides is inserted. The vector is prepared to be targeted in vivo, and appropriate host cells are transformed therewith and then, cultured to expression under a suitable condition according to the method of Sambrook, et al. (Molecular Cloning, 1989, Cold Spring Harbor, Cold Spring Harbor Laboratory Press). Also, the peptides may be prepared by use of fusion proteins containing the amino acid sequence according to the present invention.
In the present invention, the amino acid sequences of the peptides can be varied according to any conventional method known in the art. For example, the peptides can be varied by changing the number of amino acids. The peptides are also varied by substitution or conversion of specific residues except those directly involving in the binding or having to be conserved, within the scope of not deteriorating the activity of the peptides. The amino acids may be modified not only to naturally occurring L-xcex1-amino acids but also to D-xcex1-amino acids as well as xcex2, xcex3 or xcex4-amino acids.
Typically, as a result of analyzing the effects of electrostatic force or hydrophilicity on binding, the sensitivity is likely to be changed in case of substitution of positively-charged amino acids, e.g. Lys, Arg, His, or negatively-charged amino acids, e.g. Glu, Asp, Asn, Gln. As mentioned above, kind and number of residues that can be substituted or added are determined depending upon required space between the essential binding points and required functions such as hydrophilicity or hydrophobicity. By such substitution, the affinity of the peptides with target proteins can be further increased.
Substitution may accompany critical functional alterations. The selection of residues for substitution may greatly affect basic skeletal structures of the peptides by changing their electricity, hydrophobicity, or side chains or helical structures, etc. Variations greatly affecting properties of peptides, are exemplified by substitution of hydrophilic residues, e.g. serine, with hydrophobic residues, e.g. leucine, isoleucine, phenylalanine, valine or alanine, substitution of positively-charged residues, e.g. lysine, arginine or histidine, with negatively-charged residues, e.g. glutamic acid or aspartic acid, or substitution of residues having no side chain, e.g. glycine, with residues having bulky side chain.
Considering the above-described facts, the skilled person in the art can modify the specific peptides by using any conventional method within the scope of maintaining or enhancing, or not deteriorating the binding affinity with HRF and inhibitory activity on histamine release. This is construed to fall within the scope of the present invention.
The peptides of the present invention is useful in the diagnosis, prophylaxis or treatment of any HRF-associated allergic diseases, e.g. asthma, rhinitis, urticaria, anaphylaxis, allergic bronchiectasis, allergies due to foods, drugs, pollen, insects, etc., hay fever, cold urticaria, or atopic dermatitis. Since HRF is commonly detected in the blood of patients suffered from the above allergic diseases (see FIG. 22), the skilled person can easily anticipate that the peptides of the present invention are effective in diagnosis, prophylaxis or treatment of the above exemplified allergic diseases.
Accordingly, the invention provides a composition for prophylaxis or treating allergies comprising as an active ingredient the above peptides. The peptides can be administered with a daily dose of about 0.1xcx9c5 mg, preferably, 0.3xcx9c2.5 mg, per body weight of 1 kg. The present composition may be formulated into solutions or micelles and then, directly injected to human or animals. The composition can be applied by parenteral or topical administrations, preferably by intravenous, subcutaneous, endothelial or muscular injection. For this purpose, the peptides are dissolved or suspended in pharmaceutically acceptable carriers, particularly, in water-soluble carriers.
Further, the peptides of the present invention may be contained in a diagnosis kit of allergies. The diagnosis kit may comprise the HRF-binding peptides and anti-HRF monoclonal antibodies. In the test using the present kit, in case of positive blood reaction, it is decided that the subject is afflicted by allergies even in the absence of allergens. That is, since HRF is floating in the blood of LPR allergy patients, it can be determined whether or not HRF is present in the blood by use of the present kit, thereby to distinguish LPR patients. In one embodiment, the HRF-binding peptides are attached to the bottom of a container, reacted with a blood sample and then, conjugated anti-HRF monoclonal antibodies are added thereto.
Moreover, the present inventors examined whether or not HRF increases neurotransmitters release by inhibitory activity on (Na,K)ATPase in nerve cells. For this purpose, HRF is added to the culture solution of nerve cell line, art-known PC12 cells (Abu-Raya, et al., 1999), which contain secretory granules of neurotransmitters and thus, are particularly suitable for studying the regulation of catecholamine release, to measure changes in [3H]-labeled dopamine release. As a result, the inventors found that in PC12 cells, HRF dose-dependently increases basal and K+-stimulated releases in a depolarized state induced by the increase of K+. They also confirmed that the HRF-binding peptides effectively block neurotransmitters release induced by HRF in nerve cells.
As set forth above, HRF, which involves in the intracellular regulation of (Na,K)ATPase activity, stimulates neurotransmitters release by inhibitory activity on (Na,K)ATPase playing an important role in neuroactivity in nerve cells and therefore, is believed to play an important role in pathophysiological effects in nerve cells as well as brain. For this reason, the HRF-binding peptides capable of blocking the increase in neurotransmitters release by HRF are extremely useful for diagnosis, prophylaxis or treatment of various apoptosis-associated nerve diseases, e.g. cerebral apoplexy, Alzheimer""s disease, Parkinson""s diseases, etc. Therefore, according to the present invention, provided is a composition for diagnosis, prophylaxis or treatment of various apoptosis-associated nerve diseases, e.g. cerebral apoplexy, Alzheimer""s disease, Parkinson""s diseases, etc. In this case, the administration routes and dosages as mentioned above can be also applied.
Meanwhile, HRF is also called translationally controlled tumor protein. It was already known that an anti-malaria agent Artemisinin binds to malaria protein HRF (Bhisutthibhan, et al., 1998). Therefore, the peptides of the invention having the binding affinity with HRF can be employed in prophylaxis or treatment of malaria in the same manner as Artemisinin. In this case, the administration routes and dosages as mentioned above can be also applied.